Week 4 Study Questions

Question 1

The complement system can be initiated by three different pathways, name these

Answer 1

• Classical pathway
• Mannose binding lectin pathway
• Alternative pathway

Question 2

C1q can bind to molecules other than immunoglobulin, what are some of these other proteins?

Answer 2

– C-reactive protein & pentraxin-3 (inflammatory response proteins) bound to bacteria

– Phosphatidylserine, double stranded DNA or annexins on dead and dying cells

• this binding also activates the classical pathway

Question 3

MBL generally complexes with either MASP-1 or MASP-2 but not both, how does this effect the activation of MASP-2 and the location of active complexes on pathogens?

Answer 3

• MBL circulates associated with two serine proteases MASP-1 and MASP-2; most MBL are associated with only one of the MASP-1 proteins
• Upon target binding juxtaposition of MASP-2 & MASP-1 containing MBL complexes is required for MASP-1 to activate MASP-2

Question 4

What is C3(H2O) and describe why it is important?

Answer 4

• Conversion of C3 to C3(H2O) occurs in the alternative pathway
• spontaneous hydrolysis of a labile thioester bond – converting C3 to its bioactive form C3(H2O)
• Formation of C3(H2O) causes exposure of the binding site for Factor B(FB)
• C3(H2O)-bound FB is then cleaved by serine protease Factor D forming →
  fluid phase C3 convertase, C3(H2O)Bb.
• C3(H2O)-bound FB is then cleaved by serine protease Factor D forming →
  fluid phase C3 convertase, C3(H2O)Bb
• C3(H2O)Bb cleaves C3 to give C3a & C3b -> initiation of alternative pathway

Question 5

Describe how the Classical/MBL pathways are regulated at pathway initiation

Answer 5

• A plasma serine protease inhibitor, C1 inhibitor (C1 INH) controls the activation of C1 complex.
• C1 INH binds C1r:C1s causing them to dissociate from C1q, limiting the time active C1s has to cleave C4 & C2.
• C1 INH regulates the MBL pathway by inhibiting MASP-1 & MASP-2.
• MBL can also bind MASP-3 – but MASP-3 cannot activate MASPs or cleave C4 or C2

Question 6

Classical and MBL pathways regulated at C3/C5 convertase

Answer 6

• C4b2a C3 convertase on a host cell will be rapidly dissociated by DAF (decay
  accelerating factor/CD55) &/or CR1 (cell surface complement receptor-1/CD35)
  depending on the cell type, releasing C2a.
• Bound C4b will be inactivated by Factor I (FI) in the presence of co-factors CR1 and /or MCP (membrane cofactor protein/CD46)
• FI is a serine protease found in plasma – but requires co-factors for cleavage.
• C4d remains bound to the membrane but C4c is released
• C4 binding protein (C4BP), a serum protein, acts in the fluid phase and on cell surfaces.
• It can interact with several C4b molecules at once – dissociating the C2a.
• C4BP also serves as a co-factor for FI – in the presence of C4BP FI can cleave C4b to give the inactive C4d and C4c

Question 7

How is the terminal pathway regulated?

Answer 7

• Binding of C5b to cell membranes is relatively non-specific - but generally occurs adjacent to site of complement activation.
• Clusterin and vitronectin bind C8 and prevents insertion into the membrane
• CD59 (protectin) is widely distributed in mammalian cell membranes, and it inhibits the binding of C9 to the C5b678 complex → blocks MAC formation.
• C5b-9 can be removed by endocytosis or exocytosis

Question 8

Describe how C3b acts as an opsonin?

Answer 8

• Main opsonins are C3b, C4b and iC3b - bacteria coated by these opsonins are rapidly taken up and destroyed by phagocytic cells.
• Phagocytic cells express complement receptors CR1, CR3 and CR4, which have broad specificity for these opsonins
• The binding of iC3b to CR3 stimulates phagocytosis in its own right
• In vivo C3b is the main opsonin because so much of it is produced
• Binding of C3b to CR1 leads to phagocytosis when other immune mediators (e.g. C5a) have activated the macrophages

Question 9

What are the processes by which complement proteins act to enhance B-cell responses?

Answer 9

• Follicular DCs express complement receptors: CR1 binds iC3b &CR2 binds C3dg
• Follicular DCs present Ag-Ab complexes bound via CR1/CR2 to germinal centre
• B cells → B cell memory maintenance
• CR1 & CR2 main receptors on DCs for long term retention of Ag
• C3dg binds Ag bound to Ig on B cell surfaces & simultaneously binds CR2 on the B Cell → Signalling through both membrane Ig & CR2 ↑↑↑ activation of B cells
• Lower threshold for B cell activation (1000x)

Question 10

Explain, using examples, how deficiencies in complement proteins can lead to disease.

Answer 10

• Generally a genetic deficiency in a classical pathway component→ autoimmune disease, increased infections or both
• e.g. lack of C1q, C1r, C1s, C4 and C2 causes susceptibility to systemic lupus erythematosus (SLE), because ↓ ability to clear immune complexes.
• Persons lacking alternative pathway components have very high risk of recurrent pyogenic bacterial infections

Question 11

Complement proteins also have activities in other biochemical pathways explain, using the coagulation pathway as an example.

Answer 11

• Activation of the coagulation pathways (the contact system and tissue factor pathways) leads to the generation of thrombin, which elicits platelet activation and fibrin formation
• involves Kallikrein cleaves (HMWK, generating bradykinin, complement proteins including C3, C5 and factor B)
• gC1qR is a multi-ligand binding protein with high affinity for HMWK & FXII
• Binding of gC1qR to C1q activates the classical complement pathway
• C1-INH is a potent inhibitor of the classical complement pathway as well as a major regulator of the kinin system

Question 12

How does complement contribute to sepsis?

Answer 12

• In sepsis, high levels of infectious micro-organisms in the blood cause excessive complement activation, leading to ⇑ ⇑ C5a
• Blocking antibodies to C5a are protective in animal models

1. Immune depletion- paralysis of neutrophils & apoptosis of leukocytes
2. severe inflammation triggering a “cytokine storm”
3. uncontrolled coagulation
4. Neutrophil hyperactivity

• Massive complement activation causes Neutrophil dysfunction in sepsis (abnormal chemotaxis, phagocytosis, TNF etc)

Question 13

Complement activation and regulation are finely tuned. An imbalance can lead to an attack on healthy host cells and trigger immune and inflammatory diseases. What are some of these diseases and how do complement proteins contribute?

Answer 13

• Diseases that activate the alternative pathway of complement cause decreased levels of C3, but C4 levels are normal - e.g. membranoproliferative glomerulonephritis
• A deficiency of C1 inhibitor (C1INH) causes hereditary angioedema - repeated, life-threatening episodes of edema,
  especially of the skin, upper respiratory tract and gastrointestinal tract
• In disseminated intravascular coagulation, ↓ C3 is due to the digestion of C3 by enzymes involved in clot fibrinolysis. C4 levels are normal.

Question 14

Alternative pathway regulated at C3/C5 convertase

Answer 14

• MCP, DAF (CD55) and CR1 can dissociate Bb from C3bBb. Factor H (FH) is an abundant serum protein that can also displace Bb from C3bBb
• C3b with co-factors: MCP, CR1
  or FH, is rapidly cleaved to iC3B by FI.
• With Co factor CR1, FI cleaves iC3b to give C3c (released into fluid) and cell bound C3dg, which becomes C3d by cell
  proteases or plasmin

Question 15

Role of serum protein factor H in C3/C5 convertase of alternative pathway

Answer 15

• Serum protein Factor H (FH) competes with Factor B for binding to C3b in the fluid phase and on cell surfaces.
• Factor B binds C3b → alternative pathway progresses;
• Factor H binds C3b → alternative pathway does not progress.
• Surface of mammalian cells (presence of sialic acid) favours Factor H binding, whereas surface of bacteria favours Factor B binding.
• In the fluid phase Factor H binds to C3b and C3(H2O), but NOT C3